Sodalis glossinidius is a facultative intracellular bacterium that is a secondary symbiont of the tsetse fly, which carries the trypanosome that causes African sleeping sickness. Although the presence of Sodalis within the cells of the tsetse fly has been documented since the mid-1980s, the physiological processes that enable this facultative endosymbiotic bacterium to survive and multiply within the tsetse fly and the intracellular environment are just beginning to be examined. Investigating these processes will contribute to the long-term scientific goals of the PIs research, which are to identify the metabolic and physiological processes that facultative intracellular bacteria employ to survive and/or multiply within their hosts, as well as the elements that regulate gene expression in these environments. The scientific goals of this project are to test hypotheses which address the contribution of putative Sodalis iron acquisition systems to growth of Sodalis within insect cells and the tsetse fly. One system (HemR/HemTUV) is predicted to encode transport proteins that mediate acquisition of heme as an iron source. Heme should be readily available to Sodalis residing in midgut tissue since the tsetse fly blood meal is digested in the midgut to release free heme. The second system is predicted to encode an achromobactin siderophore system that mediates high affinity iron acquisition. This system may be important when Sodalis is within the tsetse fly cells and/or in other environments where free iron is in limiting quantities. In Specific Aims 1 and 2, the ability of the Sodalis heme and achromobactin transporter mutants to grow in tsetse flies and in insect culture will be examined to test hypotheses related to the question of which Sodalis iron acquisition genes are important for growth of Sodalis in these environments. In Specific Aim 3, the function of the putative Sodalis heme and achromobactin transporters will be assessed by analyzing growth of the Sodalis heme and achromobactin transporter mutants in media with either heme or ferri- achromobactin as a sole iron source and by reconstituting the Sodalis systems in heterologous bacterial strains that cannot use heme or achromobactin as iron sources. Studying the tsetse fly-Sodalis symbiosis will have significance to public health. The presence of Sodalis in the tsetse fly may increase tsetse-mediated transmission of the trypanosome, and recent research suggests that paratransgenesis may be a useful control strategy for African trypanosomiasis. For these reasons, a more complete understanding of the nature of the Sodalis-tsetse symbiosis will yield information to help control tsetse-borne disease. PUBLIC HEALTH RELEVANCE: Tsetse-transmitted trypanosomiasis represents a significant global health burden, threatening millions of people in sub-Saharan Africa. Since the presence of Sodalis in the tsetse fly may increase tsetse-mediated transmission of the trypanosome and since recent research suggests that modifying Sodalis glossinidius (one of the bacterial species harbored by the tsetse fly) may be a useful control strategy for African trypanosomiasis, a more complete understanding of the nature of the Sodalis-tsetse symbiosis will yield information to help control tsetse-borne disease. Finally, because Sodalis is one of only a few intracellular insect symbionts to be cultured outside of the host, it can be developed as a model for the study of other intracellular insect symbionts that have an impact on the ability of the insect to transmit disease.